Snežana Zdravković-Korać

Cytokinin Profiles of AtCKX2-Overexpressing Potato Plants and the Impact of Altered Cytokinin Homeostasis on Tuberization In Vitro

Genes encoding cytokinin oxidase/dehydrogenase (CKX) enzymes have been used lately to study cytokinin homeostasis in a variety of plant species. In this study AtCKX2-overexpressing potato plants were engineered and grown in vitro as a model system to investigate the effects of altered cytokinin levels on tuber formation and tuber size. Protein extracts from shoots and roots of transformed potato plants exhibited higher CKX activity compared to control plants. Total endogenous cytokinin levels were generally not decreased in AtCKX2 overexpressors. However, levels of bioactive cytokinins were markedly lowered, which was accompanied by increased levels of O- and N-glucosides in some transgenic lines. The AtCKX2-overexpressing plants displayed reduced shoot growth but other symptoms of the “cytokinin deficiency syndrome” were not recorded. The transgenic plants were able to produce tubers in noninducing conditions. In inducing conditions they developed larger tubers than control. Tubers were also formed on a greater portion of the analyzed AtCKX2 plants, but with a lower number of tubers per plant compared to control. Taken together, our data suggest that cytokinins cannot be regarded simply as positive or negative regulators of tuberization, at least in vitro. Interactions with other plant hormones that play an important role in control of tuberization, such as gibberellins, should be further studied in detail.

Pyramiding rice cystatin OCI and OCII genes in transgenic potato (Solanum tuberosum L.) for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say)

Proteinase inhibitors oryzacystatins I and II (OCI and OCII) have shown potential in controlling pests that utilize cysteine proteinases for protein digestion. In order to achieve an additive effect against Colorado potato beetle (CPB, Leptinotarsa decemlineata Say) digestive proteinases, we combined two individual OC genes in potato (Solanum tuberosum L.) cultivars Desiree, Dragačevka and Jelica. The OC genes were stacked into the potato genome by simultaneous co-transformation with both OCI and OCII genes or by sequential re-transformation of an OCI-expressing transgenic line with an OCII gene. Low constitutive and high wound-induced transcript levels of both OCI and OCII genes, directed by the inducible pin2 promoter, were detected in all doubly transformed lines from all three cultivars. Both recombinant OCs, detected by immunoblot, were in an active conformation as shown by their strong papain inhibitory activity. Although no significant differences in CPB larval mortality were observed when larvae fed on OCI/OCII transformed or control potato foliage, expression of both OCI and OCII in potato plants caused a reduction in CPB adult body weight and in weight of foliage consumed, indicating an additive effect. Overall, these results show that pyramiding of the OCI and OCII genes could be an effective strategy for breeding cultivars exhibiting elevated levels of resistance to CPB.

Use of Chenopodium murale L. transgenic hairy root in vitro culture system as a new tool for allelopathic assays

We investigated Chenopodium murale transgenic hairy root in vitro culture system as a new tool for allelopathic assays. Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS from roots, cotyledons, leaves, and internodes of C. murale seedlings. Roots were found to be the best target explants, providing transformation efficiency of up to 11.1%. Established hairy root clones differed in their morphology and growth potential. Molecular characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS analyses. No differences in rol gene expression were observed. Liquid culture system of characterized hairy root clones was maintained for over 2 years. Six hairy root clones were selected for assaying the allelopathic effect of their growth medium against germination and seedling elongation of wheat and lettuce test plants. The inhibitory potential varied depending on the hairy root clone. Some transgenic clones showed significantly higher inhibition compared to wild-type roots. These results revealed that hairy roots as an independent system synthesize some bioactive substances with allelopathic activity and exude them into the growth medium. Concentrations of caffeic, ferulic and p-coumaric acids (0.07-2.85 μmol/L) identified by HPLC analysis in the growth media were at least 1000 times lower than the inhibitory active concentration (5 mmol/L) of pure grade phenolic acids, suggesting that they have a limited role in the allelopathic phenomena of C. murale. The presented hairy root system appears to be a suitable tool for further investigation of the potential and nature of root-mediated allelopathic interference of C. murale.

Secondary somatic embryogenesis versus caulogenesis from somatic embryos of Aesculus carnea Hayne.: developmental stage impact

Somatic embryos of red horse chestnut, derived from cultures maintained through repetitive somatic embryogenesis for a few years, were subjected to induction of secondary regeneration. The embryos were divided in four classes on the basis of their size (I-1, II-5, III-10 and IV-30 mm), and sub-cultured on MS media containing 0, 1, 5 or 10 μM kinetin (Kin) or benzyladenine (BA). The pathway of secondary regeneration, somatic embryogenesis or caulogenesis, depended on the primary somatic embryo (PSE) stage of development. The embryogenic capacity declined and bud-forming capacity increased with the degree of PSE maturity. The PSE of the Classes I and II produced only secondary somatic embryos (SSE), the Class III PSE formed both SSE and adventitious buds, whereas the Class IV PSE developed almost solely adventitious buds. The process of secondary somatic embryogenesis was most effective in the Class II PSE at 5 μM BA, and the process of adventive organogenesis was most effective in the Class IV PSE at 10 μM BA. On plant growth regulator (PGR)-free medium, PSE of A. carnea followed the same pattern of adventive regeneration, as those cultured on cytokinin containing media. The cytokinins only amplified the response, in a certain range of concentrations. BA promoted bud induction at a much higher rate than Kin, while their embryogenic effect was similar.

Determination of escin content in androgenic embryos and hairy root culture of Aesculus hippocastanum.

Escin, a group of chemically related triterpenic glycosides, is widely used in commercial preparations for the treatment of venous insufficiency. Since the zygotic embryo cotyledons accumulate the highest amount of escin, it is currently extracted from the seeds of horse chestnut, Aesculus hippocastanum L. (Hippocastanaceae), on a large scale. As this material is available during only short period of the year, we studied the possibility of using plant tissue culture to obtain escin. For this purpose, the content of escin in androgenic embryos and hairy root cultures of horse chestnut was studied. Escin content was found to be dependent on the stage of androgenic embryo development and the type of phytoregulator supplemented to the nutritive medium. In the absence of phytoregulators, androgenic embryos at the globular stage of development contained approximately four times less escin than those at the cotyledonary stage. Inclusion of various phytoregulators in the nutritive media stimulated escin production. Among them, 2,4-dichlorophenoxyacetic acid (2,4-D) showed the most pronounced effect, with escin content almost reaching that found in zygotic embryos (6.77% versus 6.96%). Two hairy root clones produced substantial amounts of escin (3.57% and 4.09%), less than zygotic embryos, but higher than cotyledonary embryos on phytoregulator-free medium.

Hairy roots formation in recalcitrant-to-transform plant Chenopodium rubrum

Susceptibility of C. rubrum to Agrobacterium-mediated transformation was demonstrated by inoculating the petioles of in vitro grown plants with A. rhizogenes strain A4M70GUS. Hairy roots were produced in 8 % of explants. They were isolated and maintained on plant growth regulator-free solid or liquid half-strength Murashige and Skoog medium for two years. Hairy root fresh mass increased 30 — 90 folds when grown in liquid medium, which was superior to solid medium, where most of the hairy roots produced calli. When these calli were grown on medium supplemented with 0.5 mg dm-3 thidiazuron, embryo-like structures were obtained. Transgenic status of long-term callus and hairy root cultures was confirmed by histochemical GUS assay, by PCR specific to the uidA, rolA&B and ags genes and by Southern hybridization.

Variability in shoot cultures regenerated from hairy roots of Gentiana punctata

Differences among three clones of Gentiana punctata L. hairy root shoot regenerants were investigated in relation to their growth patterns, production of secondary metabolites and 2D protein profiles. Prominent differences in growth parameters were stable thus qualifying regenerant clones as true somaclones. Marked differences in protein spots were registered among the regenerant clones but not in comparison with the non-transformed control. Southern blot hybridization of regenerants showed the absence of rolA, B and C genes, initially present in the main hairy root lines. Orf13 and rolD were present and orf8 was missing in all three regenerant clones whereas orf3 was missing only in clone 2. Although lacking the three major rol genes, plants of regenerant clones retained characteristics of the hairy root phenotype.

Heat-induced accumulation of protein synthesis elongation factor 1A implies an important role in heat tolerance in potato.

AbstractMain conclusionPotato eukaryotic elongation factor 1A comprises multiple isoforms, some of which are heat-inducible or heat-upregulated and might be important in alleviating adverse effects of heat stress on plant productivity. Heat stress substantially reduces crop productivity worldwide, and will become more severe due to global warming. Identification of proteins involved in heat stress response may help develop varieties for heat tolerance. Eukaryotic elongation factor 1A (eEF1A) is a cytosolic, multifunctional protein that plays a central role in the elongation phase of translation. Some of the non-canonical eEF1A activities might be important in developing plant heat-stress tolerance. In this study, we investigated effects of heat stress (HS) on eEF1A expression at the protein level in potato, a highly heat vulnerable crop. Our results from both the controlled environment and the field have shown that potato eEF1A is a heat-inducible protein of 49.2-kDa with multiple isoforms (5–8). Increase in eEF1A abundance under HS can be mainly attributed to 2–3 basic polypeptides/isoforms. A significant correlation between eEF1A abundance and the potato productivity in the field was observed in two extremely hot years 2011 and 2012. Genomic Southern blot analysis indicated the existence of multiple genes encoding eEF1A in potato. Identification, isolation and utilization of heat-inducible eEF1A genes might be helpful for the development of the heat-tolerant varieties.